Peptide deformylase inhibitors as novel antibiotics

ABSTRACT

A macrocyclic peptide deformylase (PDF) inhibitor comprising a peptide or peptide mimetic having three residues, P1′, P2′, and P3′, wherein P2′ connects P1′ and P3′, wherein P1′ and P3′ each have a side chain, and wherein the side chains on P1′ and P3′ are crosslinked to form the macrocyclic PDF inhibitor. The side chains of P1′ and P3′ interact with the PDF active site, and preferably, P2′ has a side chain that interacts with a solvent. Also provided are methods of inhibiting the growth of a bacterium, the methods comprising contacting the bacterium with an anti-bacterial effective amount of the inventive macrocyclic PDF inhibitor. Additionally, a method of treating a bacterial infection in a subject comprising administering an effective amount of a macrocyclic PDF inhibitor to a subject in need of treatment. Additionally, methods of preparing macrocyclic PDF inhibitors comprising a) choosing an acyclic base molecule, having at least some PDF inhibitory activity, the acyclic base molecule having a first residue having a first side chain that interacts with the PDF active site and a second residue having a second that interacts with the PDF active site; and b) crosslinking the first side chain and the second side chain to form a macrocyclic PDF inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Utility patent application Ser. No.10/899,207, filed Jul. 26, 2004, which claims priority to ProvisionalPatent Application Ser. No. 60/490,052, filed Jul. 25, 2003, theentirety of which is incorporated herein by reference.

STATEMENT ON FEDERALLY FUNDED RESEARCH

The present invention was made, at least in part, under NationalInstitutes of Health grant AI40575. The government may have certainrights in this invention.

BACKGROUND OF THE INVENTION

The emergence of antibiotic-resistant bacteria has created an urgentdemand for new antibacterial agents with novel mechanisms of action.Peptide deformylase (PDF), an essential enzyme involved in bacterialprotein biosynthesis and maturation, is one of the few novel targetsthat are currently being pursued for antibacterial drug design.¹⁻³ Inbacteria, protein synthesis starts with an N-formylmethionine and as aresult, all newly synthesized polypeptides carry a formylatedN-terminus.⁴ PDF catalyzes the subsequent removal of the formyl groupfrom the majority of those polypeptides, many of which undergo furtherN-terminal processing by methionine aminopeptidase to produce matureproteins. As an essential activity for survival,⁵⁻⁷ PDF is present inall eubacteria. On the other hand, protein synthesis in the eukaryoticcytoplasm does not involve N-terminal formylation and PDF apparently hasno catalytic function in the mammalian mitochondrion.⁸

PDF is a unique metallopeptidase, which utilizes a ferrous ion (Fe²⁺) tocatalyze the amide bond hydrolysis.^(9,10) Due to sensitivity of theFe²⁺ center to environmental oxygen and other reactive oxygen species,native PDF is extremely unstable and difficult to work with.¹¹ However,substitution of Ni²⁺ or Co²⁺ for the Fe²⁺ ion renders the enzyme highlystable while retaining almost full catalytic activity and substratespecificity of the native enzyme. Consequently, most of the recentbiochemical, structural, and inhibition studies were carried out withthe metal-substituted forms.

Numerous PDF inhibitors have been reported in recent years; essentiallyall of them are metal chelators. On the basis of the chelator structure,they can be classified into three different types: the thiols,¹²⁻¹⁴ thehydroxamates,¹⁵⁻¹⁹ and the N-formylhydroxylamines or reversehydroxamates.^(20,21) Many of the hydroxamate and reverse hydroxamateinhibitors exhibit excellent antibacterial activities in vitro and inanimal studies. One of the reverse hydroxamates from British Biotech(BB-86398) is currently in phase I clinical trials. However, since mostof these inhibitors still have significant peptide characteristics,there are some concerns about their selectivity (e.g., inhibition ofmatrix metalloproteases) and in vivo stability (e.g., proteolysis of thepeptide bonds).

SUMMARY OF THE INVENTION

Macrocyclic peptide deformylase (PDF) inhibitors are provided. Themacrocyclic PDF inhibitor is a peptide or peptide mimetic, comprising inorder, residues P1′, P2′ and P3′, wherein P2′ connects P1′ and P3′. P1′and P3′ each have side chains, wherein the side chains on P1′ and P3′are crosslinked to form the macrocyclic PDF inhibitors; the crosslinkedside chains of P1′ and P3′ interact with the active site. P2′ preferablyhas a side chain. More preferably, the side chain on P2′ interacts withthe solvent.

The macrocyclic PDF inhibitors may be depicted by formula I:

wherein the P2′ residue is selected from the group consisting of the 20naturally occurring L-amino acids, the D-amino acids, amino acidmimetics, and unnatural amino acids wherein Z is selected from the groupconsisting of H, C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl, C₁-C₁₀ alkynyl; andwherein Z can contain any number of heteroatoms, aromatic rings, orheterocycles; Y is a metal ligand, preferably selected fromN-formylhydroxylamine, hydroxamate, sulfhydryl, and carboxyl; morepreferably, Y is selected from N-formylhydroxylamine, and hydroxamate;the crosslinked P1′ and P3′ side chains may comprise saturatedhydrocarbons, unsaturated hydrocarbons, wherein the unsaturation may beone or more carbon-carbon double bonds, carbon-carbon triple bonds, orcombinations thereof; one or more aryl groups; an arylalkyl; analkylaryl; a heterocycle; and combinations thereof; and wherein anycarbon atom in the cross-linked P1′ and P3′ side chains may be replacedwith a heteroatom selected from the group consisting of O, N, and S; andn is 1 to 13, preferably n is 1 to 8, and more preferably n is 3 to 5.

A preferred subset of macrocyclic PDF inhibitors are those of formulaII:

wherein n is 1 to 8, preferably n is 3 to 5.

Another preferred subset of macrocyclic PDF inhibitors are those offormula III:

wherein n is 1 to 8, preferably n is 3 to 5.

Another preferred subset of macrocyclic PDF inhibitors are those offormula IV:

wherein n is 1 to 8, preferably n is 3 to 5.

Also provided are methods of inhibiting the growth of a bacterium, themethod comprising contacting the bacterium with an anti-bacterialeffective amount of the macrocyclic PDF inhibitor of formula I, II, III,or IV, or a combination thereof.

Also provided are methods of treating a bacterial infection in a subjectcomprising administering an effective amount of a macrocyclic PDFinhibitor of the present invention to a subject having a bacterialinfection. This method of treating a bacterial infection is useful bothwhen the bacterial infection is a drug-sensitive bacterial infection andwhen the bacterial infection is a drug-resistant bacterial infection. Inaccordance with the present invention, preferably, the subject is ahuman subject.

Also provided are methods of preparing a stable, selective PDFinhibitor, the method comprising the steps of a) choosing an acyclicbase molecule, preferably a peptide, or peptide mimetic, having at leastsome PDF inhibitory activity, the acyclic base molecule having a firstresidue with a first side chain that interacts with the PDF active siteand a second residue with a second side chain that interacts with thePDF active site; and b) crosslinking the first side chain and the secondside chain to form a macrocyclic PDF inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Retrosynthetic analysis of a macrocyclic peptide deformylase(PDF) inhibitor.

FIG. 2 Synthetic scheme for acid 4.

FIG. 3 Synthetic scheme for compound 2.

FIG. 4 (A) Electronic absorption spectra of Co(II)-substituted PDF (240μM) in the absence and presence of inhibitor 2 (500 μM). (B) Modelshowing the binding mode of inhibitor 2 to the E. coli PDF active site.Modeling was carried out by docking compound 2 into the structure of E.coli PDF bound with reverse hydroxamate inhibitor 1 (PDB access code1G27). Protein residues involved in hydrophobic interactions with theinhibitor nonyl ring, as well as the metal ligands are labeled.

FIG. 5 Inhibition of B. subtilis (A) and E. coli (B) cell growth byinhibitor 2. An overnight culture was diluted 1000-fold into 2 mL offresh LB medium containing the specified concentrations of inhibitor 2and incubated at 37° C. Cell densities were measured at the specifiedtimes on a UV-vis spectrophotometer.

FIG. 6 HPLC tracing for compound 2 (monitored at 214 nm). The peak att_(R)=˜8 min. is DMSO.

FIG. 7 Comparison of the in vitro stability of PDF inhibitors 6 (cyclic)and 9 (acyclic) in rat plasma.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides macrocyclic peptide macrocyclic peptidedeformylase (PDF) inhibitors, and therapeutic and pharmaceutic compoundscomprising the PDF inhibitors. PDF inhibitors are both effective forinhibiting the growth of a broad spectrum of bacteria and are selectiveto bacteria, they are well-suited for mammalian use, and specificallyhuman use as antibacterial and antibiotic agents. Macrocyclic PDFinhibitors especially useful as antibiotic agents because they areeffective against both drug-sensitive and drug-resistant bacteria andthey are more stable in physiological conditions than acyclic PDFinhibitors.

The macrocyclic PDF inhibitor is a peptide or peptide mimetic,comprising in order, residues P1′, P2′ and P3′, wherein P2′ connects P1′and P3′. P1′ and P3′ each have side chains, wherein the side chains onP1′ and P3′ are crosslinked to form the macrocyclic PDF inhibitors; thecrosslinked side chains of P1′ and P3′ interact with the active site.P2′ preferably has a side chain. More preferably, the side chain on P2′interacts with the solvent. The macrocyclic PDF inhibitors may bedepicted by formula I:

wherein the P2′ residue is selected from the group consisting of the 20naturally occurring L-amino acids, the D-amino acids, amino acidmimetics, and unnatural amino acids; wherein Z is selected from thegroup consisting of H, C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl, C₁-C₁₀ alkynyl, andwherein Z can contain any number of heteroatoms, aromatic rings, orheterocycles; Y is a metal ligand, preferably selected fromN-formylhydroxylamine, hydroxamate, sulfhydryl, and carboxyl, andpreferably N-formylhydroxylamide or hydroxamate; the crosslinked P1′ andP3′ side chains may comprise a saturated hydrocarbon, an unsaturatedhydrocarbon wherein the unsaturation may be one or more carbon-carbondouble bonds, carbon-carbon triple bonds, or combinations thereof, oneor more aryl groups, an arylalkyl, an alkylaryl, a heterocycle, andcombinations thereof; and wherein any carbon atom in the cross-linkedP1′ and P3′ side chains may be replaced with a heteroatom selected fromthe group consisting of O, N, and S; and n is 1 to 13, preferably 1 to8, and more preferably 3 to 5.

There are several preferred subsets of formula I. One preferred subsetof macrocyclic PDF inhibitors are those of formula II:

wherein n is 1 to 8, and is preferably 3 to 5. Another preferred subsetof macrocyclic PDF inhibitors are those of formula III:

wherein n is 1 to 8, and preferably 3 to 5. Another preferred subset ofmacrocyclic PDF inhibitors are those of formula IV:

wherein n is 1 to 8, and preferably 3 to 5.

Also provided are methods of inhibiting the growth of a bacterium, themethod comprising contacting the bacterium with an anti-bacterialeffective amount of the macrocyclic PDF inhibitor of formula I, II, III,or IV, or a combination thereof.

The invention further comprises methods of treating a broad spectrum ofbacterial infections in a subject comprising administering an effectiveamount of a macrocyclic PDF inhibitor of the present invention to asubject having a bacterial infection. This method of treating abacterial infection is useful both when the bacterial infection is adrug-sensitive bacterial infection and when the bacterial infection is adrug-resistant bacterial infection. In accordance with the presentinvention, preferably, the subject is a human subject.

Also provided are methods of preparing a stable, selective PDFinhibitor, the method comprising the steps of a) choosing an acyclicbase molecule, having at least some PDF inhibitory activity, the acyclicbase molecule being a peptide or peptide mimetic, the acyclic basemolecule having a first residue having a side chain that interacts withthe PDF active site and a third residue having a side chain thatinteracts with the PDF active site; and b) crosslinking the side chainson the first residue and the second residue to form a macrocyclic PDFinhibitor.

The present invention comprises macrocyclic PDF inhibitors, which areuseful broad spectrum antibacterial agents, and particularly useful asbroad spectrum antibiotics. The invention further comprises apharmaceutical composition comprising a therapeutically effective amountof a macrocyclic PDF inhibitor, or a derivative or pharmaceuticallyacceptable salt thereof, in association with at least onepharmaceutically acceptable carrier, adjuvant, or diluent (collectivelyreferred to herein as “carrier materials”) and, if desired, other activeingredients. The active compounds of the present invention may beadministered by any suitable route known to those skilled in the art,preferably in the form of a pharmaceutical composition adapted to such aroute, and in a dose effective for the treatment intended. The activecompounds and composition may, for example, be administered orally,intra-vascularly, intraperitoneally, intranasal, intrabronchial,subcutaneously, intramuscularly or topically (including aerosol). Withsome subjects local administration, rather than system administration,may be preferred.

The terms “therapeutically effective” and “pharmacologically effective”are intended to qualify the amount of macrocyclic PDF inhibitor isnecessary to inhibit growth of bacteria. A therapeutically effective orpharmacologically effective amount will depend on the particularmacrocyclic inhibitor, the bacterium, as well as other factors. Atherapeutically effective or pharmacologically effective amount canreadily be determined by those skilled in the art.

The compounds of the present invention can be formulated into suitablepharmaceutical preparations such as tablets, capsules, or elixirs fororal administration or in sterile solutions or suspensions forparenteral administration. The therapeutic agents described herein canbe formulated into pharmaceutical compositions using techniques andprocedures well known in the art.

About 0.1 to 1000 mg of a macrocyclic PDF inhibitor described herein iscompounded with a physiologically acceptable vehicle, carrier,excipient, binder, preservative, stabilizer, flavor, etc., in a unitdosage form as called for by accepted pharmaceutical practice. Theamount of active substance in those compositions or preparations is suchthat a suitable dosage in the range indicated is obtained. Thecompositions can be formulated in a unit dosage form, each dosagecontaining from about 1 to about 500 mg, or about 10 to about 100 mg ofthe active ingredient. The term “unit dosage from” refers to physicallydiscrete units suitable as unitary dosages for human subjects and othermammals, each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient.

To prepare compositions, one or more of the therapeutic agents employedin the methods of the invention are mixed with a suitablepharmaceutically acceptable carrier. Upon mixing or addition of thetherapeutic agent(s), the resulting mixture may be a solution,suspension, emulsion, or the like. These may be prepared according tomethods known to those skilled in the art. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the compound in the selectedcarrier or vehicle. The effective concentration is sufficient fortreating a bacterial infection and may be empirically determined.

Pharmaceutical carriers or vehicles suitable for administration of thepresent therapeutic agents include any such carriers suitable for theparticular mode of administration. In addition, the active materials canalso be mixed with other active materials that do not impair the desiredaction, or with materials that supplement the desired action, or haveanother action. The present therapeutic agents may be formulated as thesole pharmaceutically active ingredient in the composition or may becombined with other active ingredients. Derivatives of the presenttherapeutic agents, such as salts or prodrugs, may also be used informulating effective pharmaceutical compositions.

The present therapeutic agents may be prepared with carriers thatprotect them against rapid elimination from the body, such astime-release formulations or coatings. Such carriers include controlledrelease formulations, such as, but not limited to, microencapsulateddelivery systems. The active compound can be included in thepharmaceutically acceptable carrier in an amount sufficient to exert atherapeutically useful effect in the absence of undesirable side effectson the patient treated.

The active ingredient may be administered at once, or may be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions.

Oral compositions will generally include an inert diluent or an ediblecarrier and may be compressed into tablets or enclosed in gelatincapsules. For the purpose of oral therapeutic administration, the activecompound or compounds can be incorporated with excipients and used inthe form of tablets, capsules, or troches. Pharmaceutically compatiblebinding agents and adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches, and the like can contain any ofthe following ingredients or compounds of a simnilar nature: a bindersuch as, but not limited to, gum tragacanth, acacia, corn starch, orgelatin; an excipient such as microcrystalline cellulose, starch, orlactose; a disintegrating agent such as, but not limited to, alginicacid and corn starch; a lubricant such as, but not limited to, magnesiumstearate; a glidant, such as, but not limited to, colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; and aflavoring agent such as peppermint, methyl salicylate, or fruitflavoring.

The term “subject” for purposes of treatment includes any human oranimal subject in need of antibacterial or antibiotic treatment. Besidesbeing useful for human treatment, the compounds of the present inventionare also useful for veterinary treatment of mammals, including companionanimals and farm animals, such as, but not limited to dogs, cats,horses, cows, sheep, and pigs. Preferably, subject means a human.

Where the term alkyl is used, either alone or with other terms, such ashaloalkyl or alkylaryl, it includes C₁ to C₁₀ linear or branched alkylradicals, examples include methyl, ethyl, propyl, isopropyl, butyl,tert-butyl, and so forth. The term “haloalkyl” includes C₁ to C₁₀ linearor branched alkyl radicals substituted with one or more halo radicals.The term “halo” includes radicals selected from F, Cl, Br, and I.

The term aryl, used alone or in combination with other terms such asalkylaryl, haloaryl, or haloalkylaryl, includes such aromatic radicalsas phenyl, biphenyl, and benzyl, as well as fused aryl radicals such asnaphthyl, anthryl, phenanthrenyl, fluorenyl, and indenyl and so forth.The term “aryl” also encompasses “heteroaryls,” which are aryls thathave carbon and one or more heteroatoms, such as O, N, or S in thearomatic ring. Examples of heteroaryls include indolyl, pyrrolyl, and soon. “Alkylaryl” or “arylalkyl” refers to alkyl-substituted aryl groupssuch as butylphenyl, propylphenyl, ethylphenyl, methylphenyl,3,5-dimethylphenyl, tert-butylphenyl and so forth. “Haloaryl” refers toaryl radicals in which one or more substitutable positions has beensubstituted with a halo radical, examples include fluorophenyl,4-chlorophenyl, 2,5-chlorophenyl and so forth. “Haloalkylaryl” refers toaryl radicals that have a haloalkyl substituent. Examples ofhaloalkylaryls include such radicals as bromomethylphenyl,4-bromobutylphenyl and so on. Carboxyamide refers to the group CONH₂,and sulfonamide refers to the group SO₂NH₂.

Also included in the family macrocyclic PDF inhibitors are thepharmaceutically acceptable salts thereof. The phrase “pharmaceuticallyacceptable salts” connotes salts commonly used to form alkali metalsalts and to form addition salts of free acids or free bases. The natureof the salt is not critical, provided that it is pharmaceuticallyacceptable. Suitable pharmaceutically acceptable acid addition salts ofthe macrocyclic PDF inhibitors may be prepared from an inorganic acid orfrom an organic acid. Examples of such inorganic acids are hydrochloric,hydrobromic, hydroiodic, nitric, carbonic, sulfuric, and phosphoricacid. Appropriate organic acids may be selected from aliphatic,cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, andsulfonic classes of organic acids, examples of which include formic,acetic, propionic, succinic, glycolic, gluconic, lactic, malic,tartaric, citric, ascorbic, glucoronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic,p-hydroxybenzoic, phenylacetic, mandelic, ambonic, pamoic,methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic,cyclohexylaminosulfonic, stearic, algenic, δ-hydroxybutyric, galactaric,and galacturonic acids. Suitable pharmaceutically acceptable baseaddition salts of macrocyclic PDF inhibitors include metallic salts madefrom aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.Alternatively, organic salts made from N,N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine(N-methylglucamine) and procaine may be used form base addition salts ofthe macrocyclic PDF inhibitors. All of these salts may be prepared byconventional means from the corresponding macrocyclic PDF inhibitors byreacting, for example, the appropriate acid or base with the macrocyclicPDF inhibitor.

Derivatives are intended to encompass any compounds which arestructurally related to the macrocyclic PDF inhibitors or which possessthe substantially equivalent activity, as measured by the derivative'sability to selectively inhibit growth of bacteria. By way of example,such compounds may include, but are not limited to, prodrugs thereof.Such compounds can be formed in vivo, Such as by metabolic mechanisms.

Structural studies of several PDF-inhibitor complexes^(20,24,25) haverevealed that the inhibitors are bound in an extended conformation andthe P1′ and P3′ side chains are similarly oriented. While the P2′ sidechain is extended toward solvent, the P1′ and P3′ side chains areengaged in intimate interactions with the enzyme. The P1′ side chain(usually an n-butyl group) fits into a deep hydrophobic pocket in thePDF active site. The P3′ side chain makes hydrophobic contacts with ashallow pocket near the active site as well as one face of the P1′ sidechain. It appears that covalent crosslinking of the P1′ and P3′ sidechains would be accommodated by the PDF active site. Moreover, therigidity introduced by cyclization may lock the inhibitor into thePDF-binding conformation and thus improve binding affinity as well asselectivity by preventing binding to other enzymes.

Molecular modeling indicated that the nonyl group should have thesufficient length to link the P1′ Cα carbon and the P3′ amino group,while maintaining the extended conformation of the peptide backbone.Retrosynthetic analysis (Scheme 1) shows that the macrocycle can beconveniently prepared via olefin metathesis from diene 3, which in turncan be prepared from acid 4 and amine 5. This synthetic strategy wouldallow for the convergent synthesis of macrocycles of different ringsizes by using the common acid 4 and varying the length of thealkenylamino group in 5.

Synthesis of acid 4 started from the commercially available 6-heptenoicacid (Scheme 2). A hydroxymethyl group was introduced at the C-2position using 4S-benzyloxazolidin-2-one as the chiral auxiliarygroup.²⁶ Removal of the auxiliary group by hydrolysis followed bycondensation with O-benzylhydroxylamine gave the hydroxamate 9, whichwas converted into β-lactam 10 through an intramolecular Mitsunobureaction.²⁷ Hydrolysis of the β-lactam furnished acid 11, which wassubsequently formylated at its benzyloxyamine moiety to give the acid 4.

Synthesis of compound 2 is shown in Scheme 3. Amine 13, which wasobtained from the corresponding alcohol 12, was condensed withN-Boc-tert-leucine. Treatment of the resulting amide with TFA resultedin the amine 5. Coupling of amine 5 with acid 4 gave the diene 3. Theterminal alkenes were crosslinked using Grubbs' rutheniumcatalyst^(28,29) to produce the 15-membered macrocycle 14. Theconfiguration of the ring C═C bond in this intermediate was notdetermined. Catalytic hydrogenation of 14 reduced the double bond andsimultaneously removed the benzyl group from the N-hydroxyl moiety togive the N-formylhydroxylamine 2. NMR and HPLC analyses indicated apurity of ˜96%.

Compound 2 was assayed against Co(II-substituted E. coli PDF using adehydrogenase assay.^(30,31) It acted as a potent inhibitor, with anapparent K₁ value of 0.67±0.2 nM. Thus, cyclization of the P1′ and P3′side chains renders compound 2˜10-fold more potent than the acyclicparent compound 1 (IC₅₀=7 nM)²⁰. To gain insight into the mechanism ofinhibition, the Co-PDF-inhibitor 2 complex was examined by UV-visiblespectroscopy. Binding of the inhibitor resulted in marked blue shift (by˜40 nm) and reduction in the maximum intensity of the D-D transitionbands in the absorption spectrum of the cobalt ion (FIG. 1 a). Thisresult suggests that compound 2 is directly ligated to the metal ion.The maximum absorptivity of ˜200 M⁻¹ cm⁻¹ for the PDF-inhibitor complexis consistent with a bidentate interaction between theN-formylhydroxylamine group and the metal and the formation of apenta-coordinated cobalt.³² Molecular modeling showed that inhibitor 2fits snugly in the active site of E. coli PDF (FIG. 1 b). TheN-formylhydroxylamine is coordinated with the metal ion via both oxygenatoms. There are three hydrogen bonds formed between the protein and theinhibitor: from Ile-44 backbone amide to P1′ carbonyl, from P2′ amide toGly-89 carbonyl, and from Gly-89 amide to P2′ carbonyl group. The nonylgroup is engaged in extensive hydrophobic interactions with protein sidechains including those of Ile-44, Ile-86, Glu-88, Ile-128, Cys-129, andHis-132. Overall, these interactions are very similar to those observedin the PDF-inhibitor 1 complex.²⁰

The in vitro antibacterial activity of the cyclic inhibitor was testedagainst E. coli and Bacillus subtilis, the representative Gram-negativeand Gram-positive bacteria, respectively.

FIG. 2 shows the bacterial cell growth curves in the presence of varyingconcentrations of inhibitor 2. Compound 2 exhibited potent antibacterialactivity against B. subtilis, with a minimal inhibitory concentration(MIC) of 2-4 μM (or 0.7-1.4 μg/mL). It is only moderately active againstE. coli, with an MIC of ˜32 μM (˜12 μg/mL). The lower activity againstE. coli is likely due to its inefficient permeation of the bacterialouter membrane and/or being removed from the cells by the efflux pump.

In conclusion, based on our earlier observations that the P1′ and P3′side chains of PDF inhibitors are closely packed in the PDF-inhibitorcomplex, we have developed a new class of macrocyclic PDF inhibitor bycovalently linking the two side chains. The cyclic inhibitor is highlypotent against PDF and has excellent to moderate antibacterial activityagainst both Gram-positive and Gram-negative bacteria. This resultdemonstrates that cyclization of the P1′ and P3′ side chains is a viableapproach to developing potent PDF inhibitors. Due to their more rigidstructures, cyclic inhibitors of this type may also have improvedstability and selectivity.

Based on earlier observations that the P₁′ and P₃′ side chains of PDFinhibitors are closely packed in the PDF-inhibitor complex, a new classof macrocyclic PDF inhibitors has been developed by covalently linkingthe two side chains. The ring size greatly affects the inhibitorproperties, with 15-17-membered rings as the optimal ring size. Comparedto their acyclic counterparts, the cyclic inhibitors of the optimal ringsizes show much higher potency against PDF (>20-fold), improvedstability against proteolytic degradation, and higher selectivity forPDF over other metalloproteases. To the best of our knowledge, thecyclic inhibitors rank among the most potent inhibitors reported to dateagainst the PDF enzyme. Furthermore, the inventive inhibitors have goodantibacterial activity against a wide spectrum of pathogens.

Experimental Methods

General. Cobalt(II)-substituted Escherichia coli PDF was overexpressedand purified to apparent homogeneity as previously described.³³ Formatedehydrogenase was purchased from Sigma (St. Louis, Mo.). Aeromonasaminopeptidase was purified according to literature procedure.³⁴ Allchemicals were purchased from either Sigma-Aldrich (St. Louis, Mo.) orAdvanced ChemTech (Louisville, Ky.). High-resolution mass spectrometrywas performed on a 3T FT-ICR mass spectrometer equipped with anelectrospray ionization source. Absorption spectroscopic measurementswere performed on a Perkin-Elmer Lambda 20 UV/V is spectrophotometer.

Synthesis of Macrocycle (2)

4S-Benzyl-3-(6-heptenoyl)-oxazolidin-2-one (7). To the solution of6-heptenoic acid (5.00 mL, 36.9 mmol) and triethylamine (12.2 mL, 92.3mmol) in THF (200 mL) was added dropwise pivaloyl chloride (4.43 mL,36.0 mmol) at −10° C. After addition the mixture was stirred for anotherhour. Lithium chloride (1.53 g, 36 mmol) and 2-oxazolidinone 6 (6.20 g,35 mmol) were added. After the reaction was complete (˜6 h), the solventwas evaporated and the residue was partitioned between ethyl acetate(150 mL) and a 5% sodium bicarbonate solution (50 mL). The organic layerwashed with 5% NaHCO₃ (2×40 mL) and brine (40 mL) and dried over sodiumsulfate. Flash chromatography using ethyl acetate and hexane as eluentgave 9.55 g of a colorless viscous liquid (yield 95%). ¹H NMR (400 MHz,CDCl₃) δ 7.33-7.19 (m, 5H), 5.81 (m, 1H), 5.00 (m, 2H), 4.67 (m, 1H),4.15 (m, 2H), 3.26 (dd, J=3.3, 13.3 Hz, 1H), 2.93 (m, 2H), 2.76 (dd,J=9.5, 13.3 Hz, 1H), 2.10 (m, 2H), 1.71 (m, 2H), 1.47 (m, 2H); ¹³C NMR(100 MHz, CDCl₃) δ 173.2, 153.4, 138.4, 135.4, 129.4, 128.9, 127.3,114.7, 66.2, 55.1, 37.9, 35.3, 33.5, 28.3, 23.7. HRESI-MS: C₁₇H₂₁NO₃Na⁺([M+Na]⁺), calc'd 310.1414, found 310.1421.

4S-Benzyl-3R-(2-hydroxymethyl-6-heptenoyl)-oxazolidin-2-one (8). To thesolution of 4S-benzyl-3-(6-heptenoyl)-oxazolidin-2-one 7 (2.25 g, 7.8mmol) in dichloromethane (100 mL) was added titanium chloride (1.0 M indichloromethane, 8.2 mmol). The resulting solution was stirred for 10mill at 0° C. Then diisopropylethylamine (1.50 mL, 8.6 mmol) was addedto the resulting yellow slurry, and the solution turned reddish brown.After the mixture was stirred for 30 min., parafomaldehyde (0.35 g, 11.7mmol) was added, followed by the addition of a second batch of titaniumchloride (8.2 mmol). The reaction was quenched after 2 h by the additionof a saturated ammonium chloride solution (100 mL). The organic layerwas separated and the aqueous layer was extracted with dichloromethane(2×40 mL). The combined organic phase washed with brine (2×40 mL) anddried over sodium sulfate. The crude product was purified by flashchromatography using ethyl acetate and hexane as the eluent to afford1.30 g of the desired product (52% yield). ¹H NMR (250 MHz, CDCl₃) δ7.34-7.20 (m, 5H), 5.79 (m, 1H), 4.96 (m, 2H), 4.70 (m, 1H), 4.19 (m,2H), 3.95 (m, 1H), 3.88 (m, 2H), 3.28 (dd, J=3.4, 13.5 Hz, 1H), 2.81(dd, J=9.3, 13.5 Hz, 1H), 2.23 (brs, 1H), 2.05 (m, 2H), 1.71-1.40 (m,4H); ¹³C NMR (63 MHz, CDCl₃) δ 176.2, 154.0, 138.6, 135.6, 129.9, 129.4,127.8, 115.3, 66.6, 64.3, 55.9, 45.9, 38.3, 34.1, 28.4, 26.9. HRESI-MS:C₁₈H₂₃NO₄Na⁺ ([M+Na]⁺), calcd 340.1519, found 340.1527.

N-Benzyloxy-2R-hydroxymethyl-6-heptenamide (9). To a solution ofcompound 8 (0.51 g, 1.6 mmol) in THF-H₂O (24 mL, 5:1) was added hydrogenperoxide (30% concentration, 6.4 mmol) and then lithium chloride hydrate(0.13 g, 3.2 mmol) at 0° C. After the reaction was complete (˜2 h),sodium sulfite (1.23 g, 3.2 mmol) was added and the solution was stirredfor 10 min. THF was removed under vacuum and the remaining aqueoussolution washed with dichloromethane (3×40 mL). After acidification with1 N HCl to pH˜3, the solution was extracted with ethyl acetate (3×40mL). The organic layers were combined, dried over sodium sulfate, andconcentrated to give 2R-hydroxymethyl-6-heptenoic acid (0.27 g). ¹H NMR(250 MHz, D₂O) δ 6.03 (m, 1H), 5.16 (m, 2H), 3.88 (d, J=6.0 Hz, 2H),2.78 (m, 1H), 2.23 (m, 2H), 1.76-1.53 (m, 4H); ¹³C NMR (63 MHz, D₂O)179.8, 139.6, 115.0, 62.9, 48.3, 33.2, 27.7, 26.1. The above crude acid,O-benzylhydroxylamine (0.31 g, 2.5 mmol), and triethylamine (0.28 mL,2.0 mmol) were dissolved in acetonitrile (8 mL), to which2-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU) (1.10 g, 1.7 mmol) was added at 0° C. Afterstirring for 3 h, the solution was diluted with ethyl acetate (100 mL),and washed with 1 N HCl (2×40 mL), 10% sodium bicarbonate (2×40 mL), andbrine (40 mL). The solution was dried over sodium sulfate and evaporatedto give the crude product, which was purified by silica gelchromatography (87% yield). ¹H NMR (250 MHz, CDCl₃-CD₃COCD₃) δ 9.80(brs, 1H), 7.37 (m, 5H), 5.78 (m, 1H), 4.97 (m, 4H), 3.66 (m, 2H), 2.25(brs, 1H), 2.12 (m, 2H), 1.78 (m, 1H), 1.45 (m, 3H).

N-Benzyloxy-α-(4-penten-1-yl)-β-Lactam (10). Compound 9 (1.74 g, 6.6mmol) and triphenylphosphine (4.00 g, 15.3 mmol) were dissolved in THF(150 mL) and diisopropyl azodicarboxylate (2.70 mL, 13.7 mmol) wasadded. The yellow solution was stirred overnight at room temperature.After removing the solvent, the crude product was purified by flashchromatography on a silica gel column (1.42 g, 88% yield). ¹H NMR (250MHz, CDCl₃): δ 7.37 (m, 5H), 5.76 (m, 1H), 5.01. (m, 2H), 4.93 (s, 2H),3.35 (dd, J=5.0, 4.7 Hz, 1H), 2.90 (dd, J=2.3, 4.4 Hz, 1H), 2.88 (m,1H), 2.03 (m, 2H), 1.74 (m, 1H), 1.58-1.25 (m, 3H). ¹³C NMR (63 MHz,CDCl₃) δ 167.2, 138.4, 135.7, 129.6, 129.3, 129.0, 115.4, 78.0, 51.8,45.4, 33.7, 28.4, 26.7.

2R-[(Benzyloxyamino)methyl]-6-heptenoic acid (11). To a solution ofβ-lactam 10 (1.00 g, 4.1 mmol) in isopropanol (15 mL) was added lithiumhydroxide hydrate (0.21 g, 4.9 mmol) in water (7 mL). The mixture wasstirred overnight and then washed with dichloromethane (2×15 mL). Theaqueous solution was acidified with 1 N HCl to pH 2-3 and extracted withethyl acetate (3×40 mL). The combined organic layers were washed withbrine (20 mL), dried over sodium sulfate, and concentrated to afford theβ-amino acid (0.92 g, 93% yield). ¹H NMR (250 MHz, CDCl₃): δ 8.36 (brs,2H), 7.32 (m, 5H), 5.78 (m, 1H), 4.98 (m, 2H), 4.74 (s, 2H), 3.17 (m,2H), 2.76 (11, 1H), 2.07 (m, 2H), 1.58-1.23 (m, 4H).

2R—[(N-Benzyloxy-N-formylamino)methyl]-6-heptenoic acid (4). To asolution of acid 11 (1.32 g, 5.0 mmol) in dichloromethane (20 mL) wasadded formic acid (20.0 mmol) and acetic anhydride (10.0 mmol) in anice-water bath. The mixture was allowed to warm to room temperature andstirred overnight. The volatile material was removed under vacuum togive formylated amino acid 4 in quantitative yield. ¹H NMR (400 MHz,CD₃OD) δ 8.08 (brs, 0.5H), 7.94 (brs, 0.5H), 7.35 (m, 5H), 5.74 (m, 1H),4.98-4.87 (m, 2H), 3.78 (m, 1.5H), 3.42 (m, 0.5H), 2.71 (11, 1H), 2.02(m, 2H), 1.55-1.34 (m, 4H); ¹³C NMR (63 MHz, CD₃OD) δ165.2, 161.1,139.7, 131.4, 131.2, 130.5, 130.1, 115.8, 78.7, 46.6, 44.9, 34.9, 30.6,27.7. HRESI-MS: C₁₆H₂₁NO₄Na⁺ ([M+Na]⁺), calc'd 314.1363, found 314.1375.

5-Hexen-1-ylamine (13). To a solution of 5-hexen-1-ol (5.0 mmol) andtriethylamine (5.5 mmol) in dichloromethane (20 mL) was addedmethanesulfonyl chloride (5.5 mmol) dropwise at −5° C. The mixture wascontinuously stirred until the reaction was complete (˜2 h).Dichloromethane (50 mL) was added to the mixture followed by washingwith 10% sodium bicarbonate solution (2×15 mL) and brine (15 mL). Theorganic layer was dried over sodium sulfate and concentrated to give5-hexenyl methanesulfonate, which was used next without furtherpurification. The methanesulfonyl ester (4.0 mmol) and sodium azide (10mmol) were dissolved in 8:1 DMF-H₂O (16 mL) and stirred overnight at 50°C. The mixture was allowed to cool to room temperature, diluted in 50 mLof water, and extracted with diethyl ether (3×30 mL). The ethereallayers were combined, washed with brine (2×15 mL), dried over sodiumsulfate, and concentrated in vacuo to give 5-hexenyl azide. The azide(5.0 mmol) was dissolved in diethyl ether (50 mL) and lithium aluminumhydride (5.0 mmol) was added in three portions under argon in a waterbath. The grayish suspension was vigorously stirred for ˜40 min and thenquenched by the addition of water (5 mL). Sodium hydroxide solution wasadded to dissolve the solid and the resulting solution was extractedwith diethyl ether (5×30 mL). The organic phase was dried over sodiumsulfate and concentrated to give amine 13 (57% yield from the alcohol).¹H NMR (250 MHz, D₂O): δ 6.11-5.95 (m, 1H), 5.26-5.14 (m, 2H), 3.14 (t,J=7.3 Hz, 2H), 2.25 (m, 2H), 1.87-1.75 (m, 2H), 1.67-1.58 (m, 2H).

N-(5-Hexen-1-yl)-L-tert-leucinamide (5). L-Boc-tert-leucine (0.26 g, 1.1mmol) and amine 13 (as its trifluoroacetate salt, 0.24 g, 1.1 mmol) weredissolved in a solution of dichloromethane (10 ml) and triethylamine(0.16 mL, 1.2 mmol), and then1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (0.21g, 1.1 mmol) was added. After stirring for about 2 h the mixture wasdiluted with ethyl acetate (60 mL), washed with 5% NaHCO₃ (2×40 mL) andbrine, dried over sodium sulfate, filtered and concentrated to dryness.The residue was purified by flash chromatography to give the amide (0.30g, 88%). ¹H NMR (250 MHz, CDCl₃) δ 6.73 (m, 1H), 5.77 (m, 1H), 5.48 (d,J=9.5 Hz, 1H), 4.95 (m, 2H), 3.92 (d, J=9.5 Hz, 1H), 3.31 (m, 1H), 3.12(m, 1H), 2.04 (m, 2H), 1.57-1.35 (m, 4H), 1.43 (s, 9H), 1.00 (s, 9H);¹³C NMR (63 MHz, CDCl₃) δ 171.3, 156.4, 138.7, 115.1, 79.7, 62.6, 39.6,34.7, 33.6, 29.4, 28.7, 26.6, 25.8. This amide (198 mg, 0.64 mmol) wasdissolved in dichloromethane (4 mL) and trifluoroacetic acid (2 mL) wasadded and stirred for 2 h until all of the amide was consumed. Thevolatile substances were removed under vacuum. The residue was dissolvedin ethyl acetate (50 mL), and washed with 5% NaHCO₃ (2×40 mL) and brine,dried (sodium sulfate), filtered and concentrated to give the amine 5(132 mg, 98%). ¹H NMR (400 MHz, CDCl₃) δ 6.76 (brs, 1H), 5.79 (m, 1H),4.97 (m, 2H), 3.24 (m, 2H), 3.09 (s, 1H), 2.08 (m, 2H), 1.65 (brs, 2H),1.56-1.40 (m, 4H), 1.00 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 173.3,138.4, 114.8, 64.5, 38.8, 34.0, 33.3, 29.1, 26.7, 26.3. HRESI-MS:C₁₂H₂₄N₂ONa⁺ ([M+Na]⁺), calc'd 235.1781, found 235.1787.

N^(α)-{[(2R—N-Benzyloxy-N-formylamino)methyl]-6-heptenoyl}-N-(5-hexen-1-yl)-L-tert-leucinamide(3). This compound was prepared by coupling amine 5 and acid 4 in amanner similar to that of 9 (89% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.11(brs, 0.6H), 7.85 (brs, 0.4H), 7.37 (m, 5H), 6.70 (brs, 1H), 6.60 (brs,1H), 5.74 (m, 2H), 4.95 (m, 5H), 4.78 (brs, 1H), 4.32 (d, J=9.5 Hz, 1H),3.75-3.68 (m, 1.6H), 3.30 (m, 1H), 3.08 (m, 1.4H), 2.65 (m, 1H),2.07-1.97 (m, 4H), 1.53-1.35 (m, 8H), 0.96 (s, 9H); ¹³C NMR (100 MHz,CDCl₃) δ 173.3, 170.3, 162.8, 138.3, 138.0, 134.2, 129.6, 129.1, 128.7,115.0, 114.8, 60.5, 46.1, 45.3, 44.9, 39.3, 34.4, 33.5, 33.3, 30.2,29.0, 26.6, 26.2, 26.1.

N-(3-tert-Butyl-2,5-dioxo-1,4-diaza-cyclopentadec-10-en-6-ylmethyl)-N-benzyloxy-formamide(14). To a solution of diene 3 in dichloromethane (1.0 mM) was added 7%molar equivalent of Grubb's ruthenium catalyst.³⁵ The solution wasstirred at 40° C. for ˜20 h, allowed to cool to room temperature; andconcentrated to dryness. The residue was purified by flashchromatography to give the cyclic monomer 14 (83% yield). ¹H NMR (250MHz, CDCl₃) δ 8.13 (brs, 0.6H), 7.90 (brs, 0.4H), 7.37 (m, 5H), 6.31 (m,2H), 5.44-5.23 (m, 2H), 4.94 (m, 1H), 4.79 (brs, 1H), 4.18 (m, 1H),3.76-3.59 (m, 3H), 3.10 (brs, 0.3H), 2.82 (m, 0.5H), 2.79-2.52 (m,1.2H), 2.13-1.83 (m, 4H), 1.63-1.26 (m, 8H), 0.97 (s, 4.5H), 0.95 (s,4.5H); ¹³C NMR (63 MHz, CDCl₃) δ 171.3, 131.5, 130.0, 129.6, 129.1,61.3, 54.3, 45.7, 39.6, 38.9, 34.3, 34.1, 32.1, 31.8, 31.2, 29.7, 29.4,28.5, 28.2, 27.4, 27.4, 27.1, 26.9, 26.8. HRESI-MS: C₂₆H₃₉N₃O₄Na⁺([M+Na]⁺), calc'd 480.2833, found 480.2875.

N-(3-tert-Butyl-2,5-dioxo-1,4-diaza-cyclopentadec-6-ylmethyl)-N-hydroxy-formamide(2). The cyclic compound 14 (50.0 mg) was dissolved in ethyl acetate andmethanol (1:1, 10 mL) and 20 mg of 10% Pd/C as added. The mixture wasexposed to 1 atm of hydrogen until all the starting material wasconsumed. The charcoal was removed by filtration and the filtrate wasconcentrated to give the desired compound quantitatively. ¹H NMR (250MHz, CDCl₃) δ 9.39 (brs, 1H), 8.37 (brs, 0.27H), 7.86 (brs, 0.73H), 7.40(d, J=8.7 Hz, 0.27H), 6.94 (brs, 1H), 6.57 (brs, 0.73H), 4.33 (m, 1H),3.81 (m, 2H), 3.43 (m, 1H), 2.87 (m, 2H), 1.64 (brs, 2H), 1.45-1.23 (m,16H), 0.96 (s, 9H); ¹³C NMR (63 MHz, CDCl₃) δ 175.0, 173.3, 171.2,170.9, 157.2, 61.1, 60.4, 52.7, 44.5, 38.5, 34.6, 29.8, 28.0, 27.8,27.5, 26.8, 26.5, 25.2, 21.1, 14.2. HRESI-MS: C₁₉H₃₅N₃O₄Na⁺ ([M+Na]⁺),call'd 392.2520, found 392.2504. HPLC analysis showed a purity of ˜96%(FIG. 1).

EXAMPLES 1-5

Synthesis of Additional Antibiotic Compounds 1E-5E The followingcompounds were synthesized using the same method as disclosed forcompound 2, above:

Example No. Compound No. n 1 1E 1 2 2 3 3 3E* 4 4 4E 5 5 5E 8*Compound 2 of Example 2 is the same as compound 2, discussed above.

EXAMPLES 6-7

Synthesis of Antibiotic Compounds 6E and 7E The following compounds weresynthesized using the same general methods.

Example No. Compound No. n 6 6E 3 7 7E 8

EXAMPLE 8

Synthesis of biotic Compounds 8E Compound 8E was synthesized using thesame method as compound 2.

Example No. Compound No. n 8 8E 3

COMPARATIVE EXAMPLES 9-10

Comparative compounds 9C and 10C were synthesized to compare toCompounds 1E-8E. BB-3497 was obtained from British Biotech forcomparative purposes.

EXAMPLES 11-21

PDF Assays. The PDF reaction was coupled to formate dehydrogenase(FDH).³⁶ Assay reactions (total volume of 500 μL) typically contained 50mM Mops (pH 7.0), 5 mM NAD⁺, 0.5 unit FDH, 0-120 nM inhibitor 1 or 2,and 4 nM E. coli PDF. The mixture was reinsulated on ice for 1 h. Thereaction was initiated by the addition of substrateformyl-Met-Leu-p-nitroaniline (final concentration of 200 μM) as thelast component and the reaction progress was monitored continuously at365 nm on a Perkin-Elmer Lambda-20 UV-Vis spectrophotometer. Due to thepoor activity (high K_(M) value) of FDH, the progress curves typicallyhad an early lag phase (0-30 s) before finally reaching a linear phase(steady state). The initial rates were calculated from the linear regionof the progress curves and fitted to the Michaelis-Menten equationV=(V _(max) *[S])/(K _(M)(1+[I]/K ₁)+[S])

to obtain the apparent inhibition constant (K₁). An average K₁ value of0.67±0.20 nM was obtained from multiple sets of data. Example CompoundK₁ (nM) 11 1E 14 ± 3 12 2  0.67 ± 0.20 13 3E 0.40 14 4E 0.22 15 5E 25 ±6 16 6E  3.0 ± 1.3 17 7E 36 ± 9 18 8E 12 ± 4 19 9C — 20 10C 18 ± 1 21BB-3497 ˜7

EXAMPLE 22

Aminopeptidase Assay of Compound 2 The inhibition constant was alsodetermined independently using and aminopeptidase assay.³⁷ Assayreactions (total volume of 500 μL) containing 50 mM Hepes (pH 7.0), 150mM NaCl, 8.0 nM E. coli PDF, and 0-50 nM inhibitor 2 were incubated onice for 2 h. Prior to reaction initiation, the mixture was brought toroom temperature for 10 min. The PDF reaction was then initiated by theaddition of substrate formyl-Met-Leu-p-nitronilide (final concentration110 μM) and allowed to proceed for 3 min at room temperature beforebeing quenched by heating at 95° C. for 10 min (the inactivation processis usually complete within the first 30 s). After cooling to roomtemperature, 1.0 unit of Aeromonas aminopeptidase (AAP) was added to thesolution and the mixture was incubated for 15 min at room temperature.The absorbance at 405 nm was measured on a UV-Vis spectrophotometer. Theinitial rates were fitted to the above equation to obtain the K₁ value(0.33±0.15 nM). For all end-point assay reactions, the substrate toproduct conversion was kept at <20%.

Antimicrobial Susceptibility Testing. For Escherichia coli and Bacillussubtilis, cell growth was monitored on the Lambda-20 UV/V isspectrophotometer at 600 nm. A single bacterial colony from a plateculture was grown overnight in LB media and was diluted 1000-foiled into2 mL of fresh growth medium containing 0-64 μg/mL of test agent in a5-mL glass test tube shaken at a speed of 250 rpm. Cell growth (at 37°C.) was monitored for 0-600 min.

EXAMPLES 23-30

Inhibition of Metalloproteases (MMP) Several of the inhibitors weretested for inhibition of human matrix metalloproteases, which are oftenused as indicators of inhibitor selectivity (no inhibition of MMP isdesired). Each inhibitor was tested at two different concentrations (1.0and 10 μM). The values reported are the percentage of MMP activityremaining in the presence of added inhibitor. MMP-1 MMP2 MMP-3 MMP-9Example Inhibitor 1.0 μM 10 μM 1.0 μM 10 μM 1.0 μM 10 μM 1.0 μM 10 μM 231E 100 99 90 85 97 80 92 89 24 2 93 86 96 92 97 91 95 91 25 4E — — — — —— — — 26 5E 93 92 100 100 97 96 96 94 27 7E 99 93 94 85 100 94 96 95 288E 97 87 83 80 100 92 92 92 29 9C 50 00 27 2 19 1 39 6 30 10C 50 3 66 4532 27 46 11

The cyclic compounds (1E-8E) show little inhibition of any of the MMPs,whereas the acyclic inhibitors (9C and 10C) showed substantialinhibition of all of the MMP's. The cyclic compounds are restricted tocertain conformations, which are complementary to the PDF active site(by design) but do not fit the active sites of MMP's. The acyclicinhibitors are flexible and can adopt different conformations to fit theactive sites of both PDF and MMP's.

EXAMPLES 31-35

Antibacterial Activity of PDF Inhibitors Inhibitors 2-7E were testedagainst two or more bacterial strains for antibacterial activities. Theresults are set forth below. MIC (μg/mL) Example No./Compound No.Bacteria 31/2 32/4E 33/5E 34/6E 35/7E E. coli ˜12 >24 >24 4-8 >32 B.subtilis 0.7-1.4 ˜0.2 ˜16 ˜8 >32 E. faecalis (ATCC 29212) >32 >32 32 H.influenzae (ATCC 31517) 0.5 >32 32 M. catarrhalis (ATCC 0.62 <0.031 137054) S. aureus (ATCC 29213) 16 >32 32 S. pneumoniae (ATCC 4 8 >3249619) S. pneumoniae (ATCC 6301) 4 8 >32 S. pneumoniae (ATCC 6303) 2 4>32

Molecular Modeling. The inhibitor was designed on the basis of thestructure of E. coli PDF complexed with the N-formyl-hydroxylamineinhibitor 1 (PDB access code 1G27) with the modeling program FLO/QXP.³⁸The binding site model consisted of all protein residues within 10.0 Åof inhibitor 1. In the binding site model the P1′ n-butyl and P3′ sidechains of inhibitor 1 were crosslinked with poly(methylene) linkers ofvarious lengths, followed by energy minimization with the peptidebackbone fixed. Only the molecule with a nonyl group between the P1′ Cαcarbon and the P3′ amino group (Compound 2) was able to fit in thebinding site while maintaining staggered conformations in the linker.Subsequently, the entire molecule was subjected to torsional Monte Carlodocking searches, but no lower energy binding modes were found than thedesigned one.

Stability of Inhibitors Inhibitors 6E and comparative compound 9 werechosen to test for the effect of cyclization on inhibitor stability.They both have an L-lysine as the P₂′ residue and are expected to besensitive to proteolytic degradation by trypsin-like enzymes. Bothinhibitors were incubated in rat plasma at 37° C. and aliquots werewithdrawn at various times and analyzed by LC-MS. The cyclic inhibitor(6E) was very stable under the experimental conditions, showing nodetectable degradation after 5 h (FIG. 7). In contrast, the acycliccompound (9) showed time-dependent degradation, with approximately 25%loss after 5 h. Therefore, cyclization significantly improves thestability of the inhibitors against metabolic degradation. Our attemptsto identify the degradation products by mass spectrometry failed.

All examples disclosed herein are for illustrative purposes only and arenot meant to limit the claimed invention in any way.

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1. A macrocyclic peptide deformylase (PDF) inhibitor comprising apeptide or peptide nimetic having three residues, P1′, P2′, and P3′,wherein P2′ connects P1′ and P3′, wherein P1′ and P3′ each have a sidechain, and wherein the side chains on P1′ and P3′ are crosslinked toform the macrocyclic PDF inhibitor and wherein the crosslinked sidechains of P1′ and P3′ interact with the active site.
 2. The macrocyclicPDF inhibitor of claim 1 wherein P2′ is an amino acid or an amino acidmimetic.
 3. The macrocyclic PDF inhibitor of claim 1 having formula I:

wherein the P2′ residue is selected from the group consisting of the 20naturally occurring L-amino acids; the D-amino acids, amino acidmimetics, and unnatural amino acids wherein Z is selected from the groupconsisting of H, C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl, C₁-C₁₀ alkynyl; andwherein Z optionally contains any number of heteroatoms, aromatic rings,or heterocycles; Y is a metal ligand; the crosslinked P1′ and P3′ sidechains comprise a saturated hydrocarbon; an unsaturated hydrocarbonwherein the unsaturation is one or more carbon-carbon double bonds,carbon-carbon triple bonds, or combinations thereof; one or more arylgroups; an arylalkyl; an alkylaryl; a heterocycle; and combinationsthereof; and wherein any carbon atom in the cross-linked P1′ and P3′side chains is optionally replaced with a heteroatom selected from thegroup consisting of O, N, and S; and n is 1-13.
 4. The macrocyclic PDFinhibitor of claim 3 wherein Y is selected from the group consisting ofN-formylhydroxylamine, hydroxamate, sulfhydryl, and carboxyl.
 5. Themacrocyclic PDF inhibitor of claim 4 wherein Y is selected fromN-formylhydroxylamine and hydroxamate.
 6. The macrocyclic PDF inhibitorof claim 3 wherein n is 1 to
 8. 7. The macrocyclic PDF inhibitor ofclaim 6 wherein n is 3 to
 5. 8. The macrocyclic PDF inhibitor of claim 3wherein the P2′ residue is a sulfonamide.
 9. A method of inhibiting thegrowth of a bacterium, the method comprising contacting the bacteriumwith an anti-bacterial effective amount of a macrocyclic PDF inhibitor,wherein the macrocyclic PDF inhibitor comprises a peptide or peptidemimetic having three residues, P1′, P2′, and P3′, wherein P1′ and P3′each have a side chain, wherein P2′ connects P1′ and P3′, and whereinthe side chains on P1′ and P3′ are crosslinked to form the macrocyclicPDF inhibitor.
 10. The method of claim 9 wherein the macrocyclic PDFinhibitor is of formula I:

wherein the P2′ residue is selected from the group consisting of the 20naturally occurring L-amino acids; the D-amino acids, amino acidmimetics, and unnatural amino acids wherein Z is selected from the groupconsisting of H, C₁-C₁₀alkyl, C₁-C₁₀ alkenyl, C₁-C₁₀ alkynyl; andwherein Z optionally contains any number of heteroatoms, aromatic rings,or heterocycles; Y is a metal ligand; the crosslinked P1′ and P3′ sidechains comprise a saturated hydrocarbon; an unsaturated hydrocarbonwherein the unsaturation is one or more carbon-carbon double bonds,carbon-carbon triple bonds, or combinations thereof; one or more arylgroups; an arylalkyl; an alkylaryl; a heterocycle; and combinationsthereof; and wherein any carbon atom in the cross-linked P1′ and P3′side chains is optionally replaced with a heteroatom selected from thegroup consisting of O, N, and S; and n is 1-13.
 11. The method of claim10 wherein Y is selected from the group consisting ofN-formylhydroxylamine, hydroxamate, sulfhydryl, and carboxyl.
 12. Themethod of claim 11 wherein n is 3 to
 5. 13. A method of treating abacterial infection in a subject comprising administering an effectiveamount of a macrocyclic PDF inhibitor, wherein the macrocyclic PDFinhibitor comprises a peptide or peptide mimetic having three residues,P1′, P2′, and P3′, wherein P1′ and P3′ each have a side chain, whereinP2′ connects P1′ and P3′, and wherein the side chains on P1′ and P3′ arecrosslinked to form the macrocyclic PDF inhibitor.
 14. The method ofclaim 13 wherein the macrocyclic PDF inhibitor is of formula I:

wherein the P2′ residue is selected from the group consisting of the 20naturally occurring L-amino acids; the D-amino acids, amino acidmimetics, and unnatural amino acids wherein Z is selected from the groupconsisting of H, C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl, C₁-C₁₀ alkynyl; andwherein Z optionally contains any number of heteroatoms, aromatic rings,or heterocycles; Y is a metal ligand; the crosslinked P1′ and P3′ sidechains comprise a saturated hydrocarbon; an unsaturated hydrocarbonwherein the unsaturation is one or more carbon-carbon double bonds,carbon-carbon triple bonds, or combinations thereof; one or more arylgroups; an arylalkyl; an alkylaryl; a heterocycle; and combinationsthereof; and wherein any carbon atom in the cross-linked P1′ and P3′side is optionally replaced with a heteroatom selected from the groupconsisting of O, N, and S; and n is 1-13.
 15. The method of claim 13wherein the bacterial infection is a drug-sensitive bacterial infection.16. The method of claim 13 wherein the bacterial infection is adrug-resistant bacterial infection.
 17. The method of claim 13 whereinthe subject is a human subject.
 18. A method of preparing a stable,selective PDF inhibitor, the method comprising the steps of: a) choosingan acyclic base molecule, having at least some PDF inhibitory activity,the acyclic base molecule having a first residue having a first sidechain that interacts with the PDF active site and a second residuehaving a second side chain that interacts with the PDF active site; andb) crosslinking the first side chain and the second side chain to form amacrocyclic PDF inhibitor.